Chemical substances such as, for example, hormones or neurotransmitters may bind as “primary messengers” (ligands) to the membrane of cells and thereby trigger a large variety of biochemical-physiological reactions inside these cells, which enable the latter to respond to their environment. This process is mediated by a large number of membrane-bound receptors to which ligands can bind specifically and directly. It is at the beginning of different, partly extremely complex transaction cascades. Receptors control and regulate via such cascades the activity of different cellular proteins (effector proteins). These effector proteins for their part can in turn regulate the concentration of intracellular messengers, the “secondary messengers”. Only such secondary messengers control a multiplicity of physiological reactions such as, for example, synthesis and release of hormones and neurotransmitters, cell division and cell growth and excitation and excitability of neuronal cells. Particularly important secondary messengers include cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and Ca2+ ions.
The intracellular concentration of secondary messengers is usually regulated by signal transduction cascades in which G protein-coupled receptors in the membrane of cells (GPCRs) register an extracellular signal then activate corresponding G proteins which in turn either stimulate or inhibit the activity of the corresponding effector proteins (Morris A. J. and Malbon C. C. (1999) Physiological regulation of G protein-linked signaling. Physiol. Rev., 79, 1373-1430). In addition, other proteins can modulate the activity of each individual component within the framework of a large variety of feedback mechanisms.
Deviations from the physiologically normal concentration of ligands or disruptions in the course of a signal transduction cascade, caused, for example, by the malfunction of a component involved therein, may cause severe diseases. Since GPCRs represent a particularly important interface between the extracellular and intracellular medium of the cell by serving as binding site or site of attack of a very large number of endogenous and exogenous chemical substances and, moreover, controlling in a large variety important physiological processes in virtually any vital organ or tissue, they are of outstanding interest for medical-pharmacological interventions. Particularly important areas of indication in this connection are disorders of the central and peripheral nervous system, of the cardiovascular system and the inner organs.
A therapeutic goal of the pharmaceutical industry is to develop pharmacological active compounds which activate (agonists), inhibit (antagonists) the target proteins or else modulate the activity thereof. This additionally requires detailed functional characterization of the appropriate target proteins. For this purpose, different methods have been developed in recent years, which differ, some of them markedly, with respect to their flexibility, but also to the meaningfulness of the results obtained therewith, and to their robustness and their effectiveness regarding speed, amount of work required and costs.
Now that the complementary DNA of a multiplicity of receptors have been cloned and these receptors can be expressed functionally in cell systems, the studies are carried out mainly on heterologously expressed receptors. The prior art regarding the strategies and methods used and application thereof are described, for example, in articles by Hertzberg R. P. and Pope A. J. (2000) High-throughput screening: new technology for the 21st century. Curr. Opin. Chem. Biol., 4, 445-451, Howard A. D., MacAllister G., Feighner S. D., Liu Q., Nargund R. P., van der Ploeg L. H., and Patchett A. A. (2001) Orphan G-protein-coupled receptors and natural ligand discovery. Trends Pharmacol. Sci., 22, 132-140, Civelli O., Northacker H. P., Saito Y., Wang Z., Lin S. H. and Reinscheid R. K., (2001) Novel neuro-transmitters as natural ligands of orphan G-protein-coupled receptors. Trends Neurosci., 24, 230-237, and also in the references contained therein.
More than 60% of the GPCRs known regulate the intracellular concentration of the secondary messenger cAMP. Different methods exist for studying the effect of chemical substances on such GPCRs and the corresponding effector proteins.
Some of these methods are based on direct, usually radiochemical, measurements of intracellular cAMP concentration. For this purpose, for example, the cells are stimulated, biochemically disrupted after a defined period of time and the change in cAMP concentration is determined. Although these measurement methods are very sensitive, they are usually inherently slow, cost-intensive and time-consuming. It is, moreover, not possible to monitor the change in intracellular cAMP concentration in real time. Important characteristic properties such as speed and course of an activation or inhibition can be determined only by a multiplicity of additional measurements at considerable additional expense. On the other hand, these methods are advantageous in that it is possible to study the effect of active compounds not only on GPCRs but also on the effector proteins which regulate intracellular cAMP concentration.
Another method which may be used for measuring intracellular changes in cAMP or cGMP concentration makes use of the properties of membrane-bound CNG channels. Cyclic nucleotide-gated ion channels (CNG channels) are membrane-bound proteins which have the features and properties described below (Finn J. T., Grunwald M. E. and Yau K. W. (1996) Cyclic nucleotide-gated ion channels: an extended family with diverse functions. Annu. Rev. Physio., 58, 395-426; Richards M. J. and Gordon S. E. (2000) Cooperativity and cooperation in cyclic nucleotide-gated ion channels. Biochemistry, 39, 14003-14011). CNG channels comprise (1) presumably 4 or 5 subunits (α and/or β subunits) which (2) in each case span the membrane six times and (3) possess in each case a binding site for cyclic nucleotides at the carboxy-terminal intracellular end. CNG channels are (4) activated directly and in a manner dose-dependent by cAMP or cGMP, form (5) an aqueous pore in the membrane with a conductivity which is only slightly selective for monovalent cations and are (6) likewise permeable for divalent cations such as Ca2+ ions, for example.
“Binding site for cyclic nucleotides” refers to that section in the CNG channel subunits to which the cyclic nucleotides cAMP and cGMP can bind in a dose-dependent manner. The amino acid sequence in this section determines to a considerable extent the sensitivity of a CNG channel for cAMP or cGMP (sensitivity).
“Conductivity” refers to the property of ion channels of enabling in a more or less selective manner ions to flow from the outside of the cell into the cell interior or to flow out of the cell interior to the outside. For this purpose, the ion channels form an opening in the membrane (aqueous pore), through which, depending on the state of activation of these channels, ions can flow in or out, according to the concentration gradient.
It is possible in heterologous expression systems to express functional CNG channels from identical α subunits (homooligomers; Kaupp U. B., Niidome T., Tanabe T., Terada S., Bonigk W., Stuhmer W., Cook N. J., Kanagawa K., Matsuo H., Hirose T., Miyata T. and Numa S. (1989) Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gated channel. Nature, 342, 762-766), from different a subunits (heterooligomers; Bradley J., Li J., Davidson N., Lester H. A. and Zinn K. (1994) Heteromeric olfactory cyclic nucleotide-gated channels: a subunit that confers increased sensitivity to cAMP, Proc. Natl. Acad. Sci. USA, 91, 8890-8894; Liman E. R. and Buck L. B. (1994) A second subunit of the olfactory cyclic nucleotide-gated channel confers high sensitivity to cAMP. Neuron 13, 611-621), and as heterooligomers from α and β subunits (Chan T. Y., Peng Y. W., Dhallan R. S., Ahamed B., Reed R. R. and Yau K. W. (1993) A new subunit of the cyclic nucleotide-gated cation channel in retinal rods. Nature, 362, 764-767). The β subunits alone cannot form functional channels but have exclusively modulatory functions in heterooligomeric CNG channels (Chen T. Y., Peng Y. W., Dhallan R. S., Ahamed B., Reed R. R. and Yau K. W. (1993) A new subunit of the cyclic nucleotide-gated cation channel in retinal rods. Nature, 362, 764-767). When cAMP or cGMP binds to CNG channels, these channels open in a dose-dependent manner and ions flow into the cell. Activation of the CNG channels results under physiological conditions in an increased Ca2+ conductivity of these channels and thus causes the increase in intracellular Ca2+ concentration. A change in concentration of this kind can be measured using optical Ca2+ measurement methods. Thus, these ion channels could in principle be used as cellular cAMP sensor for studying and characterizing any receptors and intracellular proteins which regulate intracellular cAMP concentration. This method is very rapid, effective and inexpensive in comparison with direct cAMP measurements. It allows a high throughput of tests per day and makes real-time measurements possible. This method is therefore in principle particularly suitable for pharmacological drug screening.
The documents U.S. Pat. No. 6,001,581 and WO 98/58074 and Gotzes F. (1995) Dissertation. ISSN 0944-2952 describe the use as cAMP sensor of CNG channels comprising the α3 subunits from the epithelium of the nose. However, such CNG channels have several decisive disadvantages when used as cellular cAMP sensors in pharmaceutical drug screening. As little as 2 μM cGMP activates these CNG channels but only 80 μM cAMP produces half-maximum activation thereof (Dhallan R. S., Yau K. W., Schrader K. A. and Reed R. R. (1990) Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons. Nature, 347, 184-187; Ludwig J., Margalit T., Eismann E., Lancet D. and Kaupp U. B. (1990) Primary structure of cAMP-gated channel from bovine olfactory epithelium. FEBS Lett. 270, 24-29). However, since intracellular cAMP concentration usually changes only by a few μM, such CNG channels are only poorly suitable as cAMP sensors, although they are suitable as cGMP sensors in principle. Moreover, even small fluctuations in intracellular cGMP concentration can interfere with the cAMP concentration measurements.
In contrast, half-maximum activation (K1/2 value) of heterooligomeric CNG channels composed of α3, α4 and β1b subunits is already obtained at a cAMP concentration of about 4 μM, while K1/2 for cGMP changes only insignificantly in comparison with the homooligomeric channels (Bonigk W., Bradley J., Muller F., Sesti F., Boekhoff I., Ronnett G. V., Kaupp U. B and Frings S., (1999) The native rat olfactory cyclic nucleotide-gated channel is composed of three distinct subunits. J. Neurosci., 19, 5332-5347. CNG channels of this kind have in principle excellent suitability as cellular cAMP sensors. Disadvantageously, however, expression of such channels in heterologous cell systems requires a lot of work and time. Moreover, small fluctuations in intracellular cGMP concentration may interfere with the cAMP sensor function.
However, it is also possible to use molecular-biological methods for preparing CNG channels which comprise only α subunits but are nevertheless highly sensitive to cAMP: in 1991, a genetically modified α3 subunit of the bovine CNG channel was described, in which subunit threonine at position 537 had been replaced with a serine (T537S) (Altenhofen W., Ludwig J., Eismann E., Kraus W., Bonigk W. and Kaupp U. B. (1991) Control of ligand specificity in cyclic nucleotide-gated channel from rod photoreceptors and olfactory epithelium. Proc. Natl. Acad. Sci. USA, 88, 9868-9872). This subunit forms CNG channels whose half-maximum activation is produced by 14 μM cAMP. Threonine T537 is located in the sequence section of the α3 subunit, which is involved to a considerable extent in binding of the cyclic nucleotides. Evidently, the amino acid in this position is particularly important for the sensitivity of the CNG channels (Altenhofen W., Ludwig J., Eismann E., Kraus W., Bonigk W. and Kaupp U. B. (1991) Control of ligand specificity in cyclic nucleotide-gated channel from rod photoreceptors and olfactory epithelium. Proc. Natl. Acad. Sci. USA, 88, 9868-9872). However, this mutation, T537S, also increases the sensitivity of the channels to cGMP (K1/2=0.7 μM). Such channels (T537S mutants) can be expressed heterologously with low expenditure, but they are, as cAMP sensor, even more susceptible to interference from small fluctuations in intracellular cGMP concentration than the heterooligomeric channels. Moreover, the sensitivity to cAMP is still not high enough in order to reliably register also small fluctuations in intracellular cAMP concentration.
Furthermore, mutations in the α3 subunit of the CNG channel are known which increase sensitivity to cAMP and additionally reduce sensitivity to cGMP. (Rich T. C., Tse T. E., Rohan D. G., Schaack J. and Karpen J. W. (2001) In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors. J. Gen. Physiol., 118; 63-78). 1.2 μM cAMP but only 12 μM cGMP produce half-maximum activation of CNG channels composed of the rat α3 subunit in which cysteine in position 460 (C460) has been replaced with tryptophan (W) and, in addition, glutamate in position 583 (E583) has been replaced with methionine (M) (C460W/E583M mutant).
It was the object of the invention to develop CNG channels as cAMP sensors whose sensitivity to cAMP or cGMP is similar to that of the C460W/E583M mutant but which are genetically modified only in one position. Such CNG channels may be used in simple and rapid cellular measuring systems efficiently and universally for pharmaceutical drug screening but also for characterizing pharmacological or potentially pharmacological target proteins.
This object is achieved by CNG channels composed of α3 subunits which have been modified in the position corresponding to threonine T537 in the bovine α3 subunit so as to have higher sensitivity to cAMP and/or higher selectivity for cAMP compared to cGMP in comparison with the wild type according to SEQ ID NO 1 and 2.
These ion channels have a sensitivity to cAMP and cGMP similar to that of the C460W/E583M mutant.
The invention moreover relates to a method for preparing these CNG channels. The invention also relates to expression vectors comprising the nucleic acids for the modified CNG channels. The invention likewise relates to cell lines which are transformed with the described expression vectors and which can express the CNG channels. Particular preference is given to cell lines capable of coexpressing heterologously either GPCRs, adenylate cyclases, phosphodiesterases or other proteins which regulate intracellular cAMP concentration together with a modified CNG channel.
The invention further relates to a method for preparing these cell lines, which comprises carrying out a transformation by means of expression vectors. According to the invention, the genes for the proteins are preferably cloned into the expression vector, followed by transformation of the cell lines.
According to the invention, preference is given to using CNG channels composed of α3 subunits. However, other subunits from bovine or other organisms are also suitable.
In the subunits used according to the invention, preference is given to replacing the amino acid corresponding to threonine at position T537 in the bovine α3 subunit with a different amino acid other than serine. Particular preference is given here to those subunits in which threonine has been replaced with methionine or valine. SEQ ID NO 3 and 4 and, respectively, SEQ ID NO 5 and 6 depict the bovine α3 subunit as an example of subunits modified in this way.
The ion channels of the invention are especially suitable as cellular cAMP sensors for measuring intracellular cAMP concentration. They are also suitable for determining the action of ligands, agonists and antagonists on G protein-coupled receptors (GPCRs) which regulate intracellular cAMP concentration. Moreover, they may be used for determining the action of activators and inhibitors on adenylate cyclases and phosphodiesterases (effector proteins) which regulate intracellular cAMP concentration.
The invention further relates to the use of cellular measuring systems comprising nucleic acids and the corresponding proteins for determining the action of chemical substances which influence the activity of cellular components which regulate intracellular cAMP concentration directly or indirectly.
The cellular measuring systems may be used universally and flexibly as cAMP sensors for pharmaceutical drug screening and drug characterization and for characterization of pharmacologically relevant proteins. The latter include all G protein-coupled receptors, adenylate cyclases, phosphodiesterases and any other proteins involved in cAMP signal pathways.
Cellular cAMP sensors which may be prepared and used instead of the T537M mutant or the C460W/E583M mutant however, are in principle also other genetically modified CNG channels which have
(i) a similarly high or higher sensitivity to cAMP,
(ii) a similarly high or higher sensitivity to cAMP or else
(iii) a similarly high or higher sensitivity to and, in addition, a similarly high or higher selectivity for cAMP.
CNG channels of this kind may have (1) α3 subunits of other organisms, (2) other CNG-channel subunits from bovine or other organisms, and (3) a homooligomeric or heterooligomeric composition of these subunits. These subunits may (4) be genetically modified in each case at the position corresponding to position T537 in the α3 subunit of the bovine CNG channel. The threonine in this position may have been replaced with a methionine or a valine or else with another amino acid (with the exception of serine). Such subunits may have (5) further genetic modifications at other positions. These CNG channels may further (6) comprise chimeric subunits which have been genetically modified in the same way at this position.
“Genetically modified at the position corresponding to position T537 in the α3 subunit of the bovine CNG channel” means the following: the different subunits of CNG channels (e.g. α1, α2, α3 and α4) or identical subunits of CNG channels of different organisms, such as, for example, the bovine and rat α3 subunits, have sequences which are highly similar to one another. Nevertheless, the position of the structurally and functionally important sections in the amino acid sequence of these subunits usually differs slightly. The skilled worker, however, is able to identify the sequence sections and amino acids corresponding to one another by comparing the sequences. Threonine T537 in the binding site for cyclic nucleotides in the bovine α3 subunit, for example, corresponds to threonine T539 in the rat α3 subunit or to threonine T560 in the bovine α1 subunit.
“Further genetic modifications” means that, in addition to a modifications of the invention, an amino acid has been replaced with a different one in at least one other position or an amino acid has been deleted from or added to at least one other position.
“Chimeric subunits” means those CNG-channel subunits which are composed of at least two different subunit moieties, i.e., for example, a subunit composed of the amino-terminal moiety of the α1 subunit and the carboxy-terminal moiety of the α3 subunit. Such chimeras can be readily prepared by a skilled worker using molecular-biological methods and are often utilized in order to combine particular properties of one protein with the properties of another protein or to transfer particular properties to another protein or else to alter particular properties in comparison with the wild-type proteins. Chimeras of different CNG-channel subunits have already been described (Seifert R., Eismann E., Ludwig J., Baumann A., and Kaupp, U. B. (1999) Molecular determinants of a Ca2+-binding site in the pore of cyclic nucleotide-gated channels: S5/S6 segments control affinity of intrapore glutamates. EMBO J., 18, 119-130).
Six genes for subunits of CNG channels are known in vertebrates (α1-α4, β1, β2). Additionally, there exist different isoforms of these subunits (Sautter A., Zonh X., Hofmann F., and Biel M. (1998) An isoform of the rod photoreceptor cyclic nucleotide-gated channel beta subunit expressed in olfactory. neurons. Proc. Natl. Acad. Sci. USA, 95, 4696-4701; Bonigk W., Bradley J., Muller F., Sesti F., Boekhoff I., Ronnett G. V., Kaupp U. B., and Frings S. (1999) The native rat olfactory cyclic nucleotide-gated channel is composed of three distinct subunits. J. Neurosci., 19, 5332-5347). The α1 subunit (Kaupp U. B., Niidome T., Tanabe T., Terada S., Bonigk W., Stuhmer W., Cook N. J., J., Kangawa K., Matsuo H., Hirose T., Miyata T., and Numa S. (1989) Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gated channel. Nature, 342, 762-766) and the β1 subunit (Chen T. Y., Peng Y. W., Dhallam R. S., Ahamed B., Reed R. R. and Yau K. W. (1993) A new subunit of the cyclic nucleotide-gated cation channel in retinal rods. Nature, 362, 764-767; Korschen H. G., Illing M., Seifert R., Sesti F., Williams A., Gotzes S., Colville C., Muller F., Dose A., Godd M., Molday L., Kaupp U. Be., and Molday R. S. (1995) A 240 kDa protein represents the complete beta subunit of the cyclic nucleotide-gated cation channel from rod photoreceptor. Neuron, 15, 627-636) were first discovered in retinal rods, the α2 subunit (Bonigk W., Altenhofen W., Muller F., Dose A., Illing M., Molday R. S., and Kaupp U. B. (1993) Rod and cone photoreceptor cells express distinct genes for cGMP-gated channels. Neuron, 10, 865-877) and the β2 subunit (Gerstner A., Zong X., Hofmann F., and Biel M. (2000) Molecular cloning and functional characterization of a new modulatory cyclic nucleotide-gated channel subunit from mouse retina. J. Neurosci., 20, 1324-1332) in retinal cones, and the α3 subunit (Dhallan R. S., Yau K. W., Schrader K. A., and Reed R. R. (1990) Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons. Nature, 347, 184-187; Ludwig J., Margalit T., Eismann E., Lancet D., and Kaupp U. B. (1990) Primary structure of cAMP-gated channel from bovine olfactory epithelium. FEBS Lett., 270, 24-29) and the α4 subunit (Bradley J., Li J., Davidson N., Lester H. A., and Zinn K. (1994) Heteromeric olfactory cyclic nucleotide-gated channels: a subunit that confers increased sensitivity to cAMP. Proc. Natl. Acad. Sci. USA, 91, 8890-8894; Liman E. R. and Buck L. B. (1994) A second subunit of the olfactory cyclic nucleotide-gated channel confers high sensitivity to cAMP. Neuron, 13, 611-621) in olfactory cells of the nose.
In addition, CNG channels composed of these subunits were found in numerous other neuronal and non-neuronal cells and tissues (Richards M. J. and Gordon S. E. (2000) Cooperativity and cooperation in cyclic nucleotide-gated ion channels. Biochemistry, 39, 14003-14011). Moreover, CNG channels were found not only in vertebrates but also in nonvertebrates such as, for example, in Drosophila melanogaster (Baumann A., Frings S., Godde M., Seifert R., and Kaupp U. B. (1994) Primary structure and functional expression of a Drosophila cyclic nucleotide-gated channel present in eyes and antennae. EMBO J., 13, 5040-5050) and plants (Leng Q., Mercier R. W., Yao W., and Berkowitz G. A. (1999) Cloning and first functional characterization of a plant cyclic nucleotide-gated cation channel. Plant Physiol., 121, 753-761). In principle, these and all other subunits of CNG channels can be modified according to the invention.
The genetically modified CNG channels may be used in cellular test systems as cAMP sensor for pharmacological studies,
(i) in order to study the action of ligands, agonists and antagonists on membrane-bound G protein-coupled receptors (GPCRs) which regulate intracellular cAMP concentration,
(ii) in order to study the action of activators and inhibitors on effector proteins (enzymes) which synthesize or hydrolyze cAMP,
(iii) in order to study the action of activators and inhibitors on other proteins which likewise intervene in the cAMP signal transduction cascade in a regulating manner, but also
(iv) in order to study the properties of GPCRs, effector proteins or other proteins involved in cAMP signal transduction cascades.
The proteins referred to as “membrane-bound G protein-coupled receptors” (GPCRs) according to the invention belong to the phylogenetically most varied, extremely extensive family of membrane-bound receptors (overview article: Morris A. J. and Malbon C. C. (1999) Physiological regulation of G protein-linked signaling. Physio. Rev., 79, 1373-1430): The family of GPCRs probably comprises distinctly more than 1,000 different members which can be classified on the basis of their sequence similarity (Probst W. C., Snyder L. A., Schuster D. I., Brosius J., and Sealfon S. C. (1992) Sequence alignment of the G-protein coupled receptor superfamily. DNA Cell Biol., 11, 1-20) or based on the chemical nature of their natural ligands. A compilation and classification of the GPCRs known up to now can be found, for example, in the “GPCRDB” database (Horn F., Weare J., Beukers M. W., Horsch S., Bairoch A., Chen W., Edvardsen O., Campagne F., and Vriend G. (1998) GPCRDB: an information system for G protein-coupled receptors. Nucleic Acids Res., 26, 275-279). Some of the representatives of the individual classes are listed below in the form of an overview. Class A (“rhodopsin-like”) includes rhodopsin itself and different sequence-related receptors which are classified on the basis of their natural ligands: these include receptors for (1) biogenic amines such as, for example, the muscarinic acetylcholine receptors, adrenergic receptors, dopamine receptors, histamine receptors, serotonin receptors, octapamine receptors, for (2) peptides, such as, for example, the angiotensin receptors, chemokine receptors, endotheline receptors, neuropeptide receptors, for (3) hormone proteins, such as, for example, FSH receptors, for (4) odorants, for (5) prostanoids, such as, for example, prostaglandin receptors, or for (6) nucleotides, such as, for example, adenosine receptors. Class B (“secretin-like”) includes the secretin receptors themselves and, for example, receptors for calcitonin, glucagon, diuretic hormones or CRF (corticotropin-releasing factor). Class C (“metabotropic glutamate/pheromones”) includes the metabotropic receptors themselves and also GABA-B receptors and others. Further classes comprise receptors from plants, fungi, insects, bacteria. All classes contain receptors whose function is not yet known or whose natural ligand is not yet known (orphan receptors). The natural ligands of each of about 200 different GPCR types are currently known and about a further 100 GPCR types are orphan GPCRs. 700 or more GPCRs are presumably activated by odorants or tastants. It is possible in principle for all GPCRs regulating intracellular cAMP concentration to be coexpressed with the inventive genetically modified CNG channels as cAMP sensor in heterologous expression systems and for the action of ligands, agonists and antagonists to be studied pharmacologically.
It is also possible to study GPCRs which normally do not regulate intracellular cAMP concentration via stimulatory or inhibitory G proteins. GPCRs of this kind may be altered by genetic modification in such a way that they couple to the cAMP signal pathway. This genetic modification may be carried out, for example, by preparing chimeric GPCRs (Liu J., Conklin B. R., Blin N., Yun J. and Wess J. (1995) Identification of a receptor-G-protein contact site critical for signaling specificity and G protein aviation. Proc. Natl. Acad. Sci. USA, 92, 11642-11646).
“Agonists” and “ligands” refer to substances which activate GCRP.
In contrast, “antagonists” refer to substances which, although being able to bind to GPCRs, cannot activate them. Antagonists inhibit the action of ligands or agonists in a dose-dependent manner.
“Effector proteins” which regulate intracellular cAMP concentration directly include adenylate cyclases and phosphodiesterases.
“Adenylate cyclases” whose activity is controlled in a GPCR-mediated manner are large, membrane-bound enzymes which catalyze the formation of cAMP from Mg.sup.2+ adenosine triphosphate and which are present in most cells, tissues and organs of the human body (Tang W. J. and Hurley J. H. (1998) Catalytic mechanism and regulation of mammalian adenylyl cyclases. Mol. Pharmacol., 54, 231-240). Nine different classes of these adenylate cyclases are known altogether. Adenylate cyclases of this kind are also endogenously expressed in the cellular test systems of the invention and can be activated by heterologously expressed GPCRs and therefore play a decisive part in the functioning of the test system. Another class comprises soluble adenylate cyclases whose activity is presumably not regulated in a GPCR-mediated manner (Buck J., Sinclair M. L., Schapal L., Cann M. J., and Levin L. R. (2000) Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals. Proc. Natl. Acad. Sci. USA, 96, 79-84). It is possible in principle to coexpress all adenylate cyclases with the inventive genetically modified CNG channels as cAMP sensor in heterologous expression systems and to study pharmacologically the action of inhibitors and activators.
“Phosphodiesterases” (PDEs) are enzymes inside the cell which hydrolyze cAMP and cGMP to give adenosine monophosphate (AMP) and guanosine monophosphate (GMP), respectively (Francis S. H., Turko I. V., and Corbin J. D. (2000) Cyclic nucleotide phosphodiesterases: relating structure and function. Prog. Nucleic Acid Res. Mol. Biol., 65, 1-52). Eleven different types of PDEs are known altogether. Some of these PDEs specifically hydrolyze cGMP, others in turn hydrolyze cAMP, and others again hydrolyze both cAMP and cGMP. Like GPCRs and adenylate cyclases, the PDEs are expressed in most cells, tissues and organs of the human body. In contrast to adenylate cyclases, however, only PDE6 which is specific for photoreceptors is activated in a GPCR-mediated manner. The activity of the other PDEs is instead regulated by different other mechanisms. It is possible in principle to coexpress all PDEs which hydrolyze cAMP with the inventive genetically modified CNG channels as cAMP sensor in heterologous expression systems and to study pharmacologically the action of inhibitors and activators.
“Activators” refers to substances which interact directly with adenylate cyclases or PDEs and thereby increase the enzymatic activity thereof.
“Inhibitors”, in contrast, refer to substances which likewise interact directly with adenylate cyclases or PDEs but which reduce the activity thereof.
It is possible in principle for all proteins which regulate intracellular cAMP concentration to be coexpressed with the inventive genetically modified CNG channels as cAMP sensor in heterologous expression systems and to be studied pharmacologically.
The proteins to be studied may be expressed in heterologous systems either transiently or, preferably, stably.
“Transient expression” means that the heterologously expressed protein is expressed by the cells of the expression system only for a defined period of time.
“Stable expression” means that the introduced gene is stably integrated into the genome of the cells of the heterologous expression system. The new cell line produced in this way expresses the corresponding protein in each subsequent cell generation.
For expression, the cDNA coding for the protein to be studied is cloned into an expression vector and transformed into the cells of a suitable expression system.
“Expression vectors” refer to any vectors which can be used for introducing (“transforming”) cDNAs into the appropriate cell lines and functionally expressing there the corresponding proteins (“heterologous expression”). Preferably, the transformation may be carried out using the pcDNA vectors (Invitrogen).
Suitable “heterologous expression systems” are in principle all eukaryotic cells such as, for example, yeast, Aspergillus, insect, vertebrate and in particular mammalian cells. Examples of suitable cell lines are CHO (Chinese hamster ovary) cells, for example the K1 line (ATCC CCL 61), including the Pro 5 variant (ATCC CRL 1781), COS cells (African green monkey), for example the CV-1 ceu line (ATCC CCL 70), including the COS-1 variant (ATCC CRL 1650) and the COS-7 variant (ATCC 1651), BHK (baby hamster kidney) cells, for example the line BHK-21 (ATCC CCL 10), MRC-5 (ATCC CCL 171), murine L cells, murine NIH/3T3 cells (ATCC CRL 1658), murine C127 cells (ATCC CRL-1616), human carcinoma cells such as, for example, the HeLa line (ATCC CCL 2), neuroblastoma cells of the lines IMR-32 (ATCC CCL 127), neuro-2A cells (ATCC CLL 131), SK-N-MC cells (ATCC HTB 10) and SK-N-SH cells (ATCC HTB 11), PC12 cells (ATCC CRL 1721), and Sf9 cells (Spodoptera frugiperda) (ATCC CRL 1711). Preference is given to using HEK 293 (human embryonic kidney) cells (ATCC CRL 1573), including the SF variant (ATCC 1573.1). Particular preference is given to the cell line prepared according to the invention, DSM ACC 2516.
The action of test substances on the protein to be studied can be measured using a fluorescence-optical measurement method.
The protein to be studied is heterologously expressed together with cAMP sensor of the invention in a cellular test system and activated or inhibited by ligands, agonists, antagonists, activators or inhibitors. The cAMP sensor registers the changes in cAMP concentration and Ca2+ ions flow into the cell to a larger or reduced extent.
“Fluorescence-optical measurement methods” means that the change in intracellular Ca2+ concentration is made visible using a fluorescent Ca2+ indicator. By now, a multiplicity of different Ca2+ indicators are known (Haugland R. P., (1996) Handbook of fluorescent probes and research chemicals. Molecular Probes Inc.). They include, for example, Fluo-3, Calcium Green, Fura-2 and Fluo-4 (Molecular Probes), the latter being preferred according to the invention. Since indicators of this kind are usually water-soluble and therefore cannot pass the hydrophobic lipid membrane of cells, the indicators are instead applied in the form of an acetoxy methyl ester compound (Tsien R. Y. (1981) A non-disruptive technique for loading calcium buffers and indicators into cell. Nature, 290, 527-528). These compounds, in contrast, are hydrophobic and are taken up by the cells. Inside the cell, the ester bond is cleaved by endogenous, intracellular esterases and the indicator is again present in its water-soluble form in which it remains in the cell interior where it accumulates and can thus be used as intracellular Ca2+ indicator. This indicator, when excited with light of a suitable wavelength, then shows fluorescence which depend on the intracellular Ca2+ concentration. The degree (amplitude) of fluorescence and the time course (kinetics) correlate with the degree and the time course of activation of the protein studied and can be monitored in real time using fluorescence detectors with a very good signal-to-noise ratio and plotted using a suitable software.
Likewise, the aequorin protein complex which consists of apoaequorin and the chromophoric cofactor coelenterazine or comparable complexes may be used as Ca2+ indicators for measuring intracellular Ca2+ concentration (Brini M., Pinton P., Pozzan T. and Rizzuto R. (1999) Targeted recombinant aequorins: tools for monitoring (Ca2+) in the different compartments of a living cell. Micrsc. Res. Tech., 46, 380-389). For this purpose, apoaequorin must be heterologously expressed together with the cAMP sensor of the invention and the protein to be studied in the cellular test system. Prior to the measurements, the cells must be incubated with coelenterazine so that apoaequorin and coelenterazine can assemble to give the active aequorin complex. When the intracellular Ca2+ concentration increases, coelenterazine is oxidized to coelenteramide. In this process, CO2 is formed and luminescence is emitted. Disadvantageously, this process is irreversible. This luminescence may be registered using a suitable optical detector (luminescence-optical measurement method). Although this optical measurement method has a similar sensitivity to the fluorescence-optical measurement method, it is however less suitable for measurements in which the course of the reaction is followed in real time.
Fluorescence- or luminescence-optical measurements using a test system of the invention may be carried out in cuvette measuring devices, in Ca2+ imaging microscopes or in fluorescence or luminescence readers.
According to the invention, preference is given to carrying out the measurements in wells of plastic containers (multiwell plates) in fluorescence readers. The cells may be introduced in suspension or else, preferably, attached to the bottom of these wells. Multiwell plates having a different number of wells may be used, such as, for example, multiwell plates having 96, 384, 1536 or more wells. These multiwell plates make it possible to carry out a multiplicity of identical or different measurements in a single plate.
“Fluorescence reader” or “luminescence reader” are very sensitive optical measuring devices which can be used to measure fluorescence or luminescence in multiwell plates. It is possible to study in such devices the action of ligands, agonists, antagonists, activators or inhibitors very rapidly and with a high throughput.
According to the invention, it is possible to study the properties of GPCRs, effector proteins or other proteins involved in cAMP signal transduction cascades quantitatively using fluorescence- or luminescence-optical measurements.
However, it is also possible in principle, according to the invention, to carry out measurements with a high throughput of tests per day. The search for new pharmacological active compounds is thus possible. It is possible here to test up to 100,000 substances per day (high throughput screening, HTS screening) or more than 100,000 substances per day (Ultra-HTS screening, UHTS screening).
Using a FLIPR384 fluorescence reader (Molecular Devices), for example, it is possible to carry out up to 384 independent measurements simultaneously and to monitor fluorescence in real time.
The invention is described in more detail below on the basis of the exemplary embodiments and the attached figures: